The invention relates generally to methods of making semiconductor nanoparticle compounds useful in the analysis of blood cell populations, and particularly to compounds which contain amino-derivatized polysaccharides.
Multiplex labeling of cells for analysis of biological samples, e.g., mixed cell populations, has been described. However, known methods have limitations which are dictated by the finite number of fluorescence emission colors of known organic fluorophores which can be squeezed into the visible, near-ultraviolet (UV), near-infrared (IR) spectral regions in which conventional measurements are made, e.g., by flow cytometry. These limitations include the widths of emission bands, the spectral overlap between these emission bands, and the excitation wavelength requirements.
Two examples of labels for cells are CdSe core nanoparticles which have been used for biological staining and observation with a fluorescence microscope [Bruchez J. M. et al., Science 281, 2013 (1998) and Chan, W. C. W. and Nie, S., Science 281, 2016 (1998)].
As the upper limit in the number of usable colors was reached, other methods, based on fluorescence intensity differences have been developed. For example, mutually exclusive pairs of targeted white blood cell populations with widely different, intrinsic numbers of receptors per cell can be labeled by a single color marker and analyzed by flow cytometry [U.S. Pat. No. 5,538,855].
Several non-radioactive gene probes, oligos with attached fluorescent dye that hybridize or bind to sample DNA have been described [L. M. Smith et al., Nature, 321:674-679 (1986) and L. M. Smith et al, Nucl. Acids Res., 13:2399-2412 (1985)] and are being used for labeling of biological samples. Automated DNA sequencers use four fluorescent dyes with non-overlapping emission bands, one per nucleotide base. However, electrophoretic mobilities of the fluorescent dye-oligo primer conjugates need to be similar for all four conjugates. Also the molecular weight of the conjugates cannot be too high, otherwise they will not move through the polyacrylamide or agarose gel used in the electrophoresis.
The need for increased sensitivity of probes used in automated analysis by attaching multiple marker molecules per oligonucleotide primer were recognized as early as 1986 [L. M. Smith et al, cited above]. However, only a limited degree of fluorescence enhancement has been possible for dye-oligo conjugates that are constrained to low molecular weight for separation by gel electrophoresis.
Aminodextrans have been used as reducing and/or protective agents in the preparation or coating of monodispersed colloidal dispersions of magnetic ferrite [U.S. Pat. No. 5,240,640], metal [U.S. Pat. No. 5,248,772], polystyrene [U.S. Pat. No. 5,466,609; U.S. Pat. No. 5,707,877; U.S. Pat. No. 5,639,620; U.S. Pat. No. 5,776,706], and polystyrene-metal [U.S. Pat. No. 5,552,086; U.S. Pat. No. 5,527,713] particles. Aminodextran of sufficiently large molecular weight can accommodate multiple protein molecules. Complexes containing such aminodextrans conjugated to a ligand and a selected fluorescent marker or label have been described. [See, Smith, C., et al, xe2x80x9cDetection of Low-Density Surface Markers Using Novel Amplified Fluorochrome-Conjugated Antibodiesxe2x80x9d, Cytometry, Suppl. 9, p. 56, presented at XIX Congress of International Society for Analytical Cytology, Mar. 3-7, 1998; R. Mylvaganam, et al., xe2x80x9cSeven Markers, Four Colors, Single Laser Flow Cytometry Using Amplified Fluorochrome Conjugated Antibodiesxe2x80x9d, Cytometry, Suppl. 9, p. 117 (1998), as presented at XIX Congress of International Society for Analytical Cytology, Mar. 3-7, 1998.] However, there continues to be a need for probes which permit increased assay sensitivity, by providing narrower fluorescence bandwidths and enhanced intensities, decreased probe size and increased probe stability.
The present invention advantageously provides particles which are of a smaller size than previously described labeled aminodextran complexes. The nanoparticles of the invention are further coated with an aminodextran of high degree of substitution to provide higher luminescence intensity than was previously possible in a single small particle.
Thus, in one aspect, the present invention provides a semiconductor nanoparticle for the analysis of fluid samples. The semiconductor nanoparticle contains a water soluble amino derivative of a polysaccharide having a molecular weight from approximately 3,000 to 3,000,000 Da, a size in diameter of less than approximately 150 nanometers, and a degree of substitution of total number of primary and secondary amino groups in the polysaccharide molecule ranging from 1/150 to 4/1. To form the semiconductor nanoparticle, this aminopolysaccharide is linked to at least one nanoparticle of the formula:
(X Y)n 
wherein X is selected from the group comprising Cd2+, Hg2+, and Zn2+ and combinations thereof; and Y is selected from the group comprising S2xe2x88x92, Se2xe2x88x92 and Te2xe2x88x92 and combinations thereof; and n=approximately 50 to 1000.
In another aspect, the present invention provides a semiconductor nanoparticle useful for the analysis of biological samples which is bound to an aminodextran having a molecular weight from approximately 3,000 to 500,000 Da, has a size in diameter of 2 to about 10 nanometers, and a degree of substitution of total number of primary and secondary amino groups in the dextran molecule ranging from 1/150 to 4/1. The aminodextran is covalently bound to at least one nanoparticle, which is defined as above.
In yet another aspect, the present invention provides a method of making a semiconductor nanoparticle. This method involves the steps of reacting an amino derivative of a polysaccharide having a molecular weight from approximately 3,000 to 3,000,000 Da with a Periodic Table Group IIB water soluble salt and a Group VIA salt to form a semiconductor nanoparticle. In this method, the semiconductor nanoparticle is a complex of the amino derivative of a polysaccharide and a nanoparticle. The aminopolysaccharide has a diameter of less than approximately 150 nanometers and a degree of substitution of total number of primary and secondary amino groups in the polysaccharide molecule ranging from 1/150 to 4/1. The Group IIB salt having a cation selected from the group consisting of Cd2+, Hg2+, and Zn2+ and combinations thereof, and an anion selected from the group consisting Clxe2x88x92, ClO4xe2x88x92, NO3xe2x88x92 and SO42xe2x88x92; said Group VIA water soluble salt having a cation selected from the group consisting of Li+, Na+, K+, Rb+, Cs+, Sr2+, and Ba2+ and an anion selected from the group consisting of S2xe2x88x92, Se2xe2x88x92 and Te2xe2x88x92 and combinations thereof; such that the anion selected for the Group IIB salt does not precipitate with the cation of the Group VIA salt. In this method, the presence of the reducing sugar in the polysaccharide retards photo-oxidation of the nanoparticle in the formed polysaccharide semiconductor nanoparticle complex.
In yet a further aspect, the present invention provides a method of making a semiconductor nanoparticle useful in the analysis of biological samples. This method involves mixing an amino derivative of a polysaccharide having a molecular weight from approximately 3,000,000 Da, has a size in diameter of less than approximately 150 nanometers, and a degree of substitution of total number of primary and secondary amino groups in the polysaccharide molecule ranging from 1/150 to 4/1, a water soluble first salt having a cation selected from the group consisting of Cd2+, Hg2+, and Zn2+ and combinations thereof, and an anion selected from the group consisting Clxe2x88x92, ClO4xe2x88x92, NO3xe2x88x92 and SO42xe2x88x92, and a water soluble second salt having a cation selected from the group consisting of Li+, Na+, K+, Rb+, Cs+, Sr2+, and Ba2+ and an anion selected from the group consisting of S2xe2x88x92, Se2xe2x88x92 and Te2xe2x88x92 and combinations thereof, such that the anion selected for the first salt does not precipitate with the cation of the second salt. Upon this mixing step, the first and second salts and the aminopolysaccharide react to form a semiconductor nanoparticle.
In yet a further aspect, the invention provides an aminopolysaccharide-semiconductor nanoparticle complex prepared according to a method of the invention. The semiconductor nanoparticle is a complex of the amino derivative of a polysaccharide and a nanoparticle.
In another aspect, the invention provides a ligand-semiconductor nanoparticle which contains a ligand conjugated to at least one semiconductor nanoparticle.
In still another aspect, the invention provides a method of preparing a ligand-semiconductor nanoparticle. The method involves mixing an amino derivative of a polysaccharide, a water soluble first salt, and a water soluble second salt to form a dispersion containing semiconductor nanoparticles. The dispersion is then purified to remove free salts, and the semiconductor nanoparticle is thereafter activated and purified. A ligand is separately activated and purified. The activated and purified ligand and semiconductor nanoparticle are then mixed, permitting formation of the ligand-semiconductor nanoparticle. The ligand-semiconductor nanoparticle is a conjugate between the selected ligand and the semiconductor nanoparticle.
In yet another aspect, the invention provides a method of detecting a target in a biological sample. The method involves contacting a biological sample suspected of containing a target for a selected ligand with a ligand-semiconductor nanoparticle of the present invention, exciting bound semiconductor nanoparticles to cause them to luminesce; and detecting the luminescence signal, thereby detecting the presence of the target in the sample.
Other aspects and advantages of the invention will be readily apparent from the detailed description of the invention.